Dear Simran,
Our standardized composition of preservation media is
For MDA MB-231: 40% FBS + 10% DMSO + 50% media.
For MCF-7: 70% FBS + 10% DMSO + 20% media.
The vials were kept in isopropanol box (Isopropanol has property to decrease temperature to -1 degree per hour, because sudden freezing can cause heavy cell mortality due to heat shock) and placed in -80 degree for 24 hours (you can keep them at -80 degree up to 1 month). Vials should be then transferred to liquid nitrogen for long term storage.
For cell revival we use 20% FBS containing complete media and maintain cells in it until cells regain their normal morphology. It’s ok to directly use cell suspension for revival (without centrifugation) if it is removed on the same day.
I think there might be problem in cell preservation, use of less concentration of cryoprotectant (DMSO) can cause "freezing injury" to cells, due to water crystal formation. Too little FBS can also be problematic for cell revival because of nutritional deprivation of cells.
I hope this might help you.....
Those dots do not look like viable cells. I am pretty certain they are not. In the second picture you can see a few cells in the top right area of the image. Those dots appear to be pieces of cells, maybe you pipetted up and down too much, maybe the freeze-down wasn't done in a desirable manner. If I thawed cells, centrifuged them out of DMSO containing media under gentle conditions, and then plated them and saw what you have taken pictures of I would thaw about 4 vials (due to the low cell density), follow thawing protocols online, and then see what you get. I'm not an expert with MCF7 cells but I have worked often with 231 cells and they are very easy to culture. Something appears to have gone wrong with your freeze-downs, handling, or growth conditions. All your cells seem to have died somewhere in the process. Hope some of this helps.
Hi Simran,
I guess they are dead, unfortunately. Are you sure that they have been frozen according to the standard protocol including 5% DMSO in the medium? DMSO also must be Biotechnology grade or similar, not technical grade, and not 20 years old.
Are you sure to follow "slow freezing" (i.e. a 2-step freezing: -80 and then liquid nitrogen) and "quick melting" protocol (from the liquid nitrogen to water bath @ 37. Carefully: the vial may explode if the nitrogen leaked into the vial because the cap has not been tighten properly!)? And of course you always transfer the melted cells into the prepared warm medium for the recovery.
Are you sure that the cells have been in a good shape before the freezing? Are you sure that you use a suitable medium? MCF7 are estrogen-dependent, while MDA-MB-231 are not.
Dear Yelena and Erik, I highly appreciate your guidance. I have thawed another 2 vials using the method Yelena has suggested (quick melting). After 4 hours, there seemed to be viable cells, so I changed the medium to remove the DMSO (as suggested by my PG supervisor). As an undergraduate, I'm not allowed to enter the lab during the weekends. I'll keep you posted on how the thawed cells turned out on Monday :) Have a good day!
Hi Simran,
I have been working on both of these cell lines since last six years. There might be a number of reasons for such issues !! It seems that your cells are not viable! Before helping you in this regard I would like to know the following....
1. Composition of preservation media for these two cell lines, as well as procedure (especially centrifugation !)
2. What was the storage temperature of frozen vials?
3. What was the percentage of FBS in cell culture media for revival of cells?
Thanks....
Dear Shivani,
1) For MDA-MB 231 (DMEM+10%FBS +5%DMSO) For MCF7 (FBS+5%DMSO)
These were quick thawed by immersing in a water bath (37C) and decanted directly in T25 flasks containing 5 mL medium (DMEM+10%FBS). My supervisor advised me not to centrifuge the cells to remove DMSO, as it may damage the cells. Instead we changed the medium after 4 hours (allowing the cells to attach to the flask).
2) The vials were stored at -80C
3) The %FBS in the media for revival was 10%.
Thank you so much for your help.
Dear Simran,
Our standardized composition of preservation media is
For MDA MB-231: 40% FBS + 10% DMSO + 50% media.
For MCF-7: 70% FBS + 10% DMSO + 20% media.
The vials were kept in isopropanol box (Isopropanol has property to decrease temperature to -1 degree per hour, because sudden freezing can cause heavy cell mortality due to heat shock) and placed in -80 degree for 24 hours (you can keep them at -80 degree up to 1 month). Vials should be then transferred to liquid nitrogen for long term storage.
For cell revival we use 20% FBS containing complete media and maintain cells in it until cells regain their normal morphology. It’s ok to directly use cell suspension for revival (without centrifugation) if it is removed on the same day.
I think there might be problem in cell preservation, use of less concentration of cryoprotectant (DMSO) can cause "freezing injury" to cells, due to water crystal formation. Too little FBS can also be problematic for cell revival because of nutritional deprivation of cells.
I hope this might help you.....
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